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Figure 6 | BMC Biochemistry

Figure 6

From: Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation

Figure 6

Stabilization of association between EF-1 complex and the 26S proteasome by phosphorylation with MPF (A) The 26S proteasome fraction from Xenopus immature oocytes was resolved by electrophoresis under denaturing conditions (12 % gel) and immunostained with anti-goldfish 26S proteasome polyclonal antibody. (B) The 26S proteasome from immature oocytes was treated with MPF for 1 hour at 30°C. Samples were treated with affinity purified anti-goldfish 26S proteasome (Anti) or control IgG (Cont) and the 26S proteasomes were immunoprecipitated. Untreated sample (Total) and precipitates were resolved by SDS-PAGE followed by autoradiography. Arrows indicate 32P-labeled 48, 37 and 30 kDa protein bands. (C) The 26S proteasome from immature oocytes was treated with (MPF+) or without (MPF-) MPF for 1 hour at 30°C. Samples were immunoprecipitated using affinity-purified anti-goldfish 26S proteasome. Supernatants (S) and precipitates (P) were immunoblotted with antibodies (α-EF-1γ; anti-recombinant EF-1γ: α-GC4/5; anti-20S proteasome α2 subunit mouse monoclonal antibody). Protein bands of EF-1γ (p48) and α2 subunit of 20S proteasome (p25) are indicated by arrows. (D) Samples were analyzed by chromatography on a G4000SWXL column equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 20% glycerol, 10 mM 2-mercaptoethanol and 0.1 mM ATP as described previously [36] in the presence of 0.5 M NaCl. Fractions were assessed by immunoblotting using a mixture of anti-Xenopus 20S proteasome and anti-EF-1γ polyclonal antibodies (α-20S+α-EF-1γ) or anti-Xenopus 20S proteasome (α-20S). Protein bands of EF-1γ (p48) and subunits of 20S proteasome are indicated by the arrow and square brackets, respectively.

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